www.nature.com/scientificreports Correction: Author Correction OPEN Fabrication of transparent hemispherical 3D nanofibrous scaffolds with radially aligned Received: 3 October 2017 patterns via a novel electrospinning Accepted: 7 February 2018 Published online: 21 February 2018 method 1 2 1,2 Jeong In Kim , Ju Yeon Kim & Chan Hee Park Tissue engineering has significantly contributed to the development of optimal treatments for individual injury sites based on their unique functional and histologic properties. Human organs and tissue have three-dimensional (3D) morphologies; for example, the morphology of the eye is a spherical shape. However, most conventional electrospinning equipment is only capable of fabricating a two-dimensional (2D) structured fibrous scaffold and no report is available on a 3D electrospinning method to fabricate a hemispherical scaffold to mimic the native properties of the cornea, including microscopic to macroscopic morphology and transparency. We proposed a novel electrospinning method using a single nonconductive hemispherical device and a metal pin. A designed peg-top shaped collector, a hemispherical nonconductive device with a metal pin in the center and copper wire forming a circle around at the edge was attached to a conventional conductive collector. A 3D hemispherical transparent scaffold with radially aligned nanofibers was successfully fabricated with the designed peg-top collector. In summary, our fabricated 3D electrospun scaffold is expected to be suitable for the treatment of injuries of ocular tissues owing to the hemispherical shape and radially aligned nanofibers which can guide the direction of the main collagen and cellular actin filament in the extracellular matrix. Recently, in the area of tissue engineering, research has focused on developing scao ff lds that can repair or replace the functions of damaged tissues or organs. A scao ff ld mimicking an extra cellular matrix (ECM) designed by 1,2 tissue engineering is used for wound sites . The ideal scao ff ld mimics the ECM structure of the target primary tissue of the target tissue . Since the ECM plays an important role in tissues and determines the survival or func- 4,5 tional maintenance of cells, studies have been carried out to imitate the cytoplasmic matrix . Supports such as a nerve conduit, an eardrum, and a cornea have been constructed by surface patterning using electrospinning 6,7 to induce cell alignment . Electrospinning has played an important role in the development of nanofibrous scaffolds for clinical use such as in tissue engineering. Polymer solutions or polymers are forced through an electric field which then elongates the polymer droplet, resulting in the formation of uniform fibers with nano to micrometer-scale diameters. In particular, biomaterials for corneal tissue engineering must demonstrate several important functions for 8–11 their potential utility in vivo, including transparency, biocompatibility and slow biodegradability . A typical treatment for injured corneal tissue is an implantation of a patch of amniotic membrane in which the cornea cells or stem cells are cultured . However, since it is difficult to obtain a human amniotic membrane (hAM) for therapeutic use, attention has been paid to developing an alternative carrier having the immune-privileged, anti-inflammatory, and growth-promoting properties of amniotic membrane for the ocular surface reconstruc - tion. Various polymeric materials have attracted interest as an alternative to this biological amniotic membrane due to their ability to meet specific needs while being able to be mass produced. Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju, 561–756, Republic of Korea. Division of Mechanical Design Engineering, College of Engineering, Chonbuk National University, Jeonju, 561-756, Republic of Korea. Correspondence and requests for materials should be addressed to C.H.P. (email: firstname.lastname@example.org) SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 1 www.nature.com/scientificreports/ Figure 1. Illustration of the shape comparison of 3D and 2D nanob fi rous scao ff lds and experimental process. In the field of materials science, various applicable biomaterials have been identified, ranging from synthetic polymers to natural polymers, which are highly soluble, inexpensive, easy to process, and biocompatible. While native polymers show better cell attachment and compatibility, they have poor mechanical properties. Therefore, functionalized electrospun fibers with improved properties need to be fabricated in combination with synthetic polymers. Among these electrospun fibers, polycaprolactone (PCL) is thermoplastic polyester with hydropho- 13,14 bic and semi-crystalline properties, which has been approved by the FDA for human medical applications . Because of the advantageous properties of PCL, it has been researched in a number of studies involving mixtures with various other polymers. Natural materials, collagen which has excellent biocompatibility and biodegradability have been extensively utilized for the manufacturing of corneal scao ff ld . The human cornea with a thickness of about 500 µm is com- posed of the stroma with its keratocytes and aligned collagen fibers . Connon et al. have confirmed that ordered collagen b fi rils and b fi ers shown to be denser, thicker, and having a higher mechanical strength than that of ran- domly oriented constituents . Also, Lindsay et al. proposed an electrospun scao ff ld containing collagen I, which replicated the unique arrangement, alignment, and morphological cues of the fibers of collagen I in the native corneal tissue . In this study, PCL and collagen nanofibrous mats have been designed and characterized to meet these func- tional requirements. The ultimate goal of this study was to fabricate an appropriate replacement for cadaveric corneas and amniotic membranes to overcome the shortage of biological membranes for transplantation. 3D nanofibrous scaffolds with radially aligned patterns have been used in biomimetic approaches to replicate the 18,19 corneal tissue structure . In the conventional electrospinning method, a 2D nanob fi rous scao ff ld can be pro- duced, but it has limitations in producing a 3D nanob fi rous scao ff ld suitable for the tissue having a curvature such as that of an eyeball (Fig. 1). The development of an electrospinning system capable of producing customizable patterned nanob fi rous 3D scao ff lds for hemispherical ocular tissue reconstruction remains a challenge . In this experiment, 3D hemispherical nanob fi rous mats with radially patterned nanob fi ers were fabricated using a mod- ified rotating collector. e Th surface of the scao ff ld radially patterned with nanob fi ers induced cell alignment. Pore sizes of 0.5–8.0 μm were introduced to the PCL/collagen nanob fi rous mat to promote the interlayer diffusion of 21–23 nutrients and to promote cell-cell interactions . The 3D nanofibrous scao ff lds with radially aligned patterns were analyzed to determine the mechanical properties, transparency, contact angle, and nanotopography using field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared spectroscopy (FT-IR) to determine the chemical and physical characteristics. The cell attachment, proliferation, and specific gene marker expression for rabbit corneal cells (rCCs) were determined by FE-SEM, Cell Counting Kit-8 (CCK-8) assay, and immunouo fl rescence. The 3D membrane was examined to verify its transparency aer t ft ransplantation, and was easily handled due to the surrounding random nanofibers for transplantation into the eyes. Combined with its ability to support corneal cell function, this optical transparency and surface pattern features of the nanob fi er mat 24–26 enable this new biomaterial system to provide significant potential benefits for corneal tissue regeneration . es Th e results indicate that the 3D nanob fi rous scao ff lds with radially aligned patterns could be suitable substitutes for corneal grafts for ocular tissue reconstruction. Materials and Methods Materials. PCL pellets and rat tail collagen purchased from Sigma-Aldrich were used to fabricate 3D radi- ally patterned nanob fi ers. 5 g of PCL pellets was stirred with a solvent of 45 g 1,1,1,3,3,3-Hexauo fl ro-2-propanol (HFIP) to prepare a 10 wt% PCL solution. Two hours prior to electrospinning, rat tail collagen (5 ml) was dis- solved in PCL solution (20 ml) and used for electrospinning. Copper wire, a metal pin, and a hemispherical non- conductor used for the modification of the rotating collector were purchased from CosmoTech and a 20-gauge syringe needle and 12 ml syringe required for electrospinning were purchased from NORM-JECT . SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 2 www.nature.com/scientificreports/ Figure 2. (A) Electrospinning setup of the fabrication of 3D radially oriented nanob fi rous scao ff lds, (B) copper wires, and metal pin in the hemispherical device designed to be electrically connected to each other. Fabrication of a 3D radially patterned nanofibrous scaffold. PCL/collagen solution for the electros- pinning process was prepared and the solution was magnetically stirred in a 10 ml vial for 12 hours. e p Th repared polymer solution was electrospun at an applied voltage of 15 kV, a tip-collector distance of 15 cm, and a solution feed rate of 1 ml/h at room temperature (25 °C). Using simple and inexpensive equipment such as copper wire and pin, we obtained a 3D radially patterned nanob fi rous scao ff ld by inducing the change of an electric field between a rotating collector and a needle, as shown in Fig. 2. First, a rotating collector was wrapped with a polyethylene sheet with copper wires at intervals of 10 cm, and hemispherical nonconductor devices with a metal pin at the center were then attached to the copper wires. The copper wires serve to supply electricity to the metal pins in hemispherical insulated conductors. The modified collector was rotated at 1000 rpm while the electrospinning was performed, and the electrospinning was performed for a total of 10 hours. The collected nanofiber mat was dried in a vacuum oven at 40 °C for one day. Electric field analysis using COMSOL . e sim Th ulation of the electric field measurement of this novel electrospinning set-up was carried out using the COMSOL Ver.4.3 added to an AC/DC module under the Window Vista operational system. The simulation of this electrospinning method was performed using the actual configuration as shown in Fig. 3. COMSOL enables analysis of the electric field and yields the results of the elec- tric field distribution simulation from the front or top view. Fiber alignment analysis via FFT and Image J. The alignment of the nanofibers was obtained using 27,28 a fast Fourier transform (FFT) method and the images were further analyzed using J software . The fiber alignment angle of the presented SEM image was converted to an output image with grayscale pixels with nano- topographical patterns . Since nanob fi ers are radially patterned, we divided the representative SEM images and confirmed the alignment of the nanob fi ers in the divided images using the FFT method. The FFT analysis data of the compartmentalized image showed the alignment of the radially patterned nanob fi ers. Characterization. e m Th orphology of the electrospun nanob fi ers of the PCL/collagen nanob fi rous mat was observed using SEM (Hitachi S-7400, Hitachi, Japan) and FE-SEM (Hitachi S-7400, Hitachi, Japan). Transparency was evaluated by performing the analysis spectra using a SYNERGY Mx spectrophotometer (BioTekR, USA) in the wavelength range of from 400 nm to 800 nm. The presence of rat tail collagen in the PCL nanofibers was determined by FT-IR spectroscopy (ABB Bomen MB100 spectrometer, Bomen, Canada) and the mechanical properties of the samples were determined using a universal tester (AG-5000G, Shimadzu, Japan) at room tem- perature. To determine the wettability of the 3D nanofibrous scao ff lds with radially aligned patterns, the water contact angle of the samples was measured using a contact angle meter (GBX, Digidrop, France) with deionized (DI) water. The experiment was conducted at a room condition and at different time intervals of 5, 10, and 15 s for a total of 8 times per sample. Biological assessment. Isolation of rCCs and culture. All experiment procedures were carried out with the approval of Chonbuk National University Animal Care Committee, Jeonju, South Korea. All experiments were performed in accordance with relevant guidelines and regulations. Two female New Zealand white rab- bits weighing 450 g were used in this experiment. The rabbits were anesthetized intramuscularly using 5cc of a mixture of Dormitor (1 mg/kg, Orion, Finland) and Alfaxalone (4 mg/kg, Jurox, Australia) for each rabbit. The SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 3 www.nature.com/scientificreports/ Figure 3. (A) Setup of the fabrication of the 3D nanob fi rous scao ff lds with radially aligned patterns (B) result of the electric field distribution simulation (front view): surface of electric field (C) contour (D) distance variation of electric field intensity (front view): horizontal line. eyes were extracted and the surrounding tissue was removed and washed in phosphate-buffered saline (PBS). e Th rabbit eye was washed three times with PBS and the cornea was peeled from the rabbit cornea. Collagenase A (0.2%, Roche, Germany) was used for digesting the cornea with Descemet’s membrane (DM) in an incubator at 37 °C for 1 h and the digested solution with the medium was centrifuged at 1500 rpm for 5 min. rCCs were suspended in medium containing epidermal growth factor, vascular endothelial growth factor, fibroblast growth factor (Clonetics, United States), hydrocortisone, gentamicin, amphotericin-B, and 10% fetal bovine serum (FBS). Cell morphology and viability. A cell culture experiment could be the first step for testing the biocompatibility 30,31 of natural or modified materials . Isolated corneal epithelial cells, endothelial cells, corneal keratocytes, and limbal stem cells have been used as a testing protocol in ophthalmological applications . In this experiment, 3D nanob fi rous PCL and PCL/collagen mats with specific nano-patterns were studied as potential matrices for ocu- lar tissue reconstruction. Their attachment and biocompatibility properties were investigated using the isolated rCCs. In addition, the impact of nanofiber orientation on the cell migration and growth were investigated. The PCL and PCL/collagen electrospun mats were cut into squares (12 mm in diameter) and sterilized for one day under UV irradiation. A 12 mm diameter cell culture plate (SPL Bioscience, Korea) in 48 wells was wrapped in a sterilized nanob fi rous mat. The corneal cells isolated from the rabbit eyes were seeded on PCL and PCL/collagen nanob fi rous mats at a density of 2 × 10 cells per well in the culture medium of 400 μl of Dulbecco Modified Eagle Medium (DMEM). Cells were inoculated in DMEM high glucose medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO . Fresh medium was added every 2 days to all samples. Aer c ft ulturing for 1, 3, and 5 days, the cell morphology was observed using SEM and a confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany). The biocompatibility was then confirmed by the CCK assay. The CCK solution (60 μl) was added to each well and cultured at 37 °C for 2 hours in an incubator. In each sample, a mixed solution of 100 μl of the cell culture medium and CCK solution was added to a 96-well plate, and the absorbance was confirmed at 450 nm using a microplate reader (Tecan, Austria). The results were presented as the mean ± standard error of the mean. Migration test. e s Th amples (random and radially aligned fibrous membranes) were cut into squares (12 mm in diameter) and sterilized for 24 hours under UV irradiation. A 12 mm diameter cell culture plate was wrapped in a sterilized electrospun fibrous membrane. The rCCs were seeded on PCL and PCL/collagen fibrous membranes at a density of 2 × 10 cells per well in the culture medium of 400 μl of DMEM high glucose medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C. After culturing for 3 days, the scratch test was adapted to electrospun membranes with a 5 × 3 × 3mm stainless steel strip for cell migration studies along their surface. Confocal laser-scanning microscopy (LSM 510 META, Carl Zeiss, Germany) were used for observing cell migra- tion aer c ft ulture for 3, 4, 5, 6, and 8 days. SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 4 www.nature.com/scientificreports/ DNA quantic fi ation assay. e m Th easurement of cellular growth in terms of DNA content was determined using TM TM a Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, USA). The DNA assay is based on the meas- urement of the fluorescence of Picogreen which is a nucleic acid strain for quantitating double-stranded DNA (dsDNA) in solution. The 10 μl of lysate was mixed with 190 μl of Pico Green in TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.5) and incubated for 5 min at room temperature, protected from light. Fluorescence was meas- ured with a SYNERGY Mx spectrophotometer (BioTekR, USA) in black 96-well cell culture plates with excitation wavelength at 480 nm and emission wavelength at 520 nm. The samples with cells were harvested aer 1, 3, a ft nd 5 days to evaluate the construct cellularity, that was assessed by determining the DNA content. The amount of cell DNA on each sample was expressed in ng/cm and the assay was repeated using a dilution of the sample to confirm the quantitation results. In-vitro Immunohistochemical examinations. For in-vitro immunohistochemical analysis, the cultured rCCs were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature and the samples were incubated for 10 min with PBS containing 0.1% Triton X-100. 1 M Quenching solution was added to the samples for 15 min for permeabilization of the cells. Aer wa ft shing the rCCs in PBS several times for 5 min, a protein blocking solution (DAKO) was added for 12 min in a dark room at room temperature. After nonspecific blocking, all samples were incubated with anti-zona ocludin-1 (ZO-1) (1:100, Santa Crux Biotechnology) as a primary antibody for 90 min at room temperature. Alexa Fluor 594-conjugated AffiniPure Donkey Anti-mouse IgG (1:250, Santa Crux Biotechnology, USA) was used for ZO-1 detection. Finally, all samples were mounted with mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Santa Crux Biotechnology, USA) and immunouo fl rescence images were obtained using a confocal LSM. mRNA Expression. Total ribonucleic acid (RNA) was extracted from rCCs cultured for 1, 3, and 5 days on control group(TCP), random, and radially aligned fibrous membranes. The rCCs cultured on the samples were TM washed with PBS and treated with the total RNA isolation solution (RiboEx , GeneAll, Korea). Extracted RNA samples were quantified using an Eppendorf BioSpectrometer (Eppendorf, Germany). Expression of mRNAs of the samples was confirmed by related genes such as ZO-1, Na+ /K+-ATPase (NaK), and chloride channel protein 3 (CLCN3). Every samples were denatured for 30 s at 95 °C and elongated for 1 min per 1 kb at 72 °C. Products of the PCR were separated by electrophoresis at 100 V on a 0.7% agarose gel (Lonza, Korea) in a 0.5% TAE buffer (Showa Chemical, Korea) and visualized using ethidium bromide (Sigma-Aldrich, Korea). Statistical analysis. Data are presented as a mean ± standard error of the mean and analyzed by one-way ANOVA. A p < 0.05 was taken as statistically significant. Results and Discussion Electric field analysis. The electric fields are created by electric charges or varying magnetic fields. Polymeric b fi ers are fabricated by a charged polymer jet oriented at external electric fields in an electrospinning process. Highly aligned or customizable patterned nanob fi ers and nanob fi rous mats can be produced by manipu- 28,29 lating electric fields . In this study, the nanob fi ers were collected using a hemispherical 3D device with a metal pin and were then radially ordered because of the adjustment of the electric fields between the needle tip and the rotating collector. The electric field simulation of the novel electrospinning was carried out using COMSOL Ver. 4.3, as shown in Fig. 3. e Th simulation was performed using the actual configuration of this electrospinning method as presented in Fig. 3A. The COMSOL program enables analysis of the electric fields from the front view, the representative distribution of the electric fields on the surface, and from a contour whereby the direction is denoted by a colored scale bar as shown in Fig. 3B and C. In addition, Fig. 3D shows the effect of the distance variation of electric field intensity (front view) and the figure is analyzed from the horizontal line in order to quantitatively analyze the results. Due to the metal pin in the hemispherical 3d shaped conductor, the electric e fi ld is most prominent in the center of the scao ff ld, which helps the nanob fi ers collect in a radially aligned orientation. The electric field on the metal pin is represented in red in Fig. 3B–D. Due to the difference in the electric field between the metal pin and the circular periphery, the nanob fi ers can be stretched and aligned, which enables the 3D nanob fi rous scao ff lds to be fabricated with radially aligned patterns . These results clearly demonstrate that the electric field is distributed and a successful 3D nanob fi rous mat with radially aligned patterns is obtained. Characterization of 3D radially oriented nanofibrous scaffold. Figure 2 shows the setup for the fab- rication of a 3D radially oriented nanob fi rous scao ff ld. The nanotopography of the nanob fi ers can be controlled by simply changing the electrospinning setup such as the applied voltage, solution flow rate, and modification of 28,29,32 the collector . In this experiment, due to the modified rotating collector, the electric field between the needle tip and the collector was changed, thereby making it possible to manufacture 3D radially oriented nanofibers. Copper wires and pins in the hemispherical device attached to the rotating collector are designed to be electrically connected to each other, as shown in Fig. 2B. SEM images of the electrospun scao ff ld fabricated in this electrospinning setup are shown in Fig. 3A–D. It was found that the nanofibers fabricated on the hemispherical equipment are radially arranged, except for the por - tion of the metal pin (Fig. 4C). On the other hand, random nanob fi ers were collected on the metal pin (Fig. 4D). Alignment analysis of the nanob fi ers by FFT reveals that radially and circumferentially aligned fibers were suc- cessfully produced as presented in Figs. 4E–I. The radially aligned nanob fi ers produce ordered gray pixels; how- ever, the randomly oriented nanob fi ers produce symmetrically distributed pixels in the output image. The shape of the peaks in the plot also reflects the degree of alignment of the nanofibers in the sample. As the degree of alignment of the nanob fi ers in the sample increased, the peaks became sharper and more pronounced compared to those in the graph of the random nanob fi ers. Radially-oriented nanob fi ers were produced by an electric field SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 5 www.nature.com/scientificreports/ Figure 4. (A,B) SEM images of radially oriented aligned nanob fi ers on the (A) and (B) parts shown in the illustration. (C) SEM image of aligned nanob fi ers on the C part shown in the illustration. (D) SEM image of random nanob fi ers on the D part shown in the illustration. (E–I) FFT output images for the nanob fi ers with pixel intensity plots against the angle of acquisition of each part of the letter, shown on the 3D radially aligned nanob fi rous mat. formed using an outer, non-conductive hemispherical device and a metal center pin. Unlike a conventional elec- trospinning setup for the fabrication of a 2D nanob fi rous mat, the electrospinning method is capable of producing a hemispherical scao ff ld such as a contact lens and is suitable for application to a curved tissue such as that of the eye, elbow, or 3D wound site. In addition, conventional 3D electrospinning has a limitation whereby it is difficult to pattern the internal nanob fi ers . The relaxed and fully stretched nanofibrous scao ff ld, as shown in Fig. 5A, confirmed that the fabricated 3D PCL/collagen nanofibros mat has sufficient mechanical strength as a corneal or wound dressing scaffold. The mechanical testing was carried out using a universal testing machine at room temperature condition and the speed head testing was set at 5 mm/min. Figure 5B shows the stress-strain curves of the 2D random nanob fi rous mat and 3D radially aligned nanofibers with a random nanofibrous mat under tensile loading. Two samples showed a comparatively linear region with a slope in the initial stress-strain curve. The toughness and elasticity of the mats significantly differed, as shown in Fig. 5C and D. The toughness of the 2D randomly oriented nano- b fi rous mat and the 3D radially aligned nanob fi rous mats was 372 ± 30 N/m and 97 ± 20 N/m, respectively. e Th elasticity of the 2D randomly oriented nanob fi rous mat and 3D radially aligned nanob fi rous mat were 13 ± 1 MPa and 11 ± 1 MPa, respectively. The 2D randomly oriented nanob fi rous mat showed a higher mechanical strength relative to the 3D radially aligned nanofibrous mat. An electrospun membrane consisting of aligned or radially aligned nanofibers has lower mechanical strength because of their fewer contact points between the fibers than an electrospun fibrous mat made of randomly oriented nanob fi ers. A lower mechanical properties were observed and reported in the aligned or radially aligned fibrous constructs regardless of the kinds of polymer. It is chal- lenging not only to fabricate a neat membrane or hemispherical 3D form with aligned nanob fi ers but also to use this for surgical applications as a corneal scao ff ld. Also, the corneal scao ff ld should have transparency, but if the aligned or radially aligned nanob fi ers are thickened to increase the mechanical strength, the transparency will be lost. For this reason, it is important to develop a method of manufacturing a corneal scao ff ld having both adequate SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 6 www.nature.com/scientificreports/ Figure 5. (A) Radially aligned nanob fi rous mat with relaxed (left) and fully stretched (right). (B ) Representative stress-strain curves of the radially aligned PCL/collagen mats. (C,D) Toughness and elasticity of PCL/collagen mats (radially aligned and randomly oriented nanob fi rous mats), showing the single mat of a radially aligned nanob fi rous mat in the form of a 3D hemisphere. (E ) Digital photographs of an eye without the scao ff ld and an eye with the scao ff ld (inset: digital photographs of cornea and a mat. The letters confirm the transparency of the radially aligned nanob fi rous mat). (F ) Transparency results using spectrum analysis method in the wavelength range of 400 nm–800 nm. (G) FT-IR spectra of the PCL and PCL/collagen nanob fi rous mats. (H ) Water contact angle plot of the PCL and PCL/collagen nanob fi rous mats. SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 7 www.nature.com/scientificreports/ mechanical strength and transparency. The radially aligned 3D scaffold developed through this study has a higher mechanical strength than the previously developed aligned nanob fi rous scao ff ld and native cornea tissue, since the periphery of the 3D radially aligned scao ff ld is connected to a membrane made of random nanob fi ers. The mechanical property of the acellular pig cornea after incubating in PBS for 1 h has studied by Kong et al. e Th proposed 3D radially aligned scao ff ld can also be seen that the mechanical strength is good enough to be used as a corneal scao ff ld when compared to the physical properties of the native corneal tissue. Transparency is an important factor for a cornea scao ff ld, not only for monitoring the process of healing but 34–36 also for the recovery of the patient’s vision ae ft r implantation . The transparency of the radially oriented PCL nanob fi rous mats blended with collagen was analyzed using a spectrophotometer in the wavelength range from 400 to 800 nm. The optical intensity of the cornea was about 0.1 in the wavelength range from 400 to 800 nm. As shown in Fig. 5F, the prepared scao ff ld was similar to the transparency of the cornea when wetted with PBS solu- tion. Thus, the transparency of the radially oriented nanob fi rous scao ff lds has an acceptable value for the cornea scao ff ld for implantation. In addition, transparency was visually confirmed through photographs of the eye and the eye implanted with scao ff lds, as shown in Fig. 5E. The scao ff ld also has good transparency, even when seen with the naked eye. FT-IR spectroscopy was used to analyze the presence of collagen in the electrospun PCL nanofibers and the −1 change in the chemical structure of the collagen. The graph in Fig. 5G shows a band of 3000–3300 cm indicating the stretching of the hydroxyl group and NH groups of the collagen groups. In addition, a band was identified at −1 −1 1634 cm , typical of the vibration mode of amide I groups and the third band at 1543 cm is attributed to the amide II due to collagen immobilization. The results of the PCL/collagen peak intensity indicated that collagen was uniformly distributed throughout the PCL nanob fi ers, as shown in Fig. 5G. Cell attachment to the scao ff ld, which is essential for cell migration, proliferation, and differentiation, can be greatly ae ff cted by the chemical composition or the physical surface architecture of the scao ff lds. The measure- ment of the contact angle can be an important assessment for the evaluation of cell attachment when the cells are seeded on the scao ff lds. The contact angle between the scao ff ld and the substrate was measured at 5, 10, and 15 s in the experiment. The time dependency of the water contact angle of the scao ff ld can influence the initial stage of the cell attachment aer ce ft ll seeding because the seeded cells can adhere more rapidly on the scao ff ld where it absorbs the cell adhesion molecules (CAM) . The contact angles of the randomly oriented and radially aligned PCL/collagen nanob fi rous mat were measured in the experiment; the average contact angles ae ft r 10 seconds were 97.5 and 94.7, respectively. The measurements showed higher contact angles for the PCL nanofibrous mat. The increase of the contact angle could be attributed to the increased porosity or the shape of the pores of the nanob fi - ers. This result shows that the aligned nano-patterned surface modification of the scao ff lds can be advantageous 37,38 for the production of scao ff lds with high contact angles . Cell proliferation. e Th topographical characteristics of the corneal scao ff lds are crucial for cell interaction and the proliferation rate of the corneal cells . The FE-SEM images in Fig. 4 show the surface topography of the samples without cell seeding. The scao ff lds constituted of aligned nanob fi ers mimicking the native ECM by pro- viding a 3D nano-pattern to induce cell growth or tissue regeneration. According to previous studies, aligned or radially oriented scao ff lds provide signic fi ant potential benet fi s for corneal regeneration and in situ correction of the corneal stroma. The morphology of corneal cells on the samples was evaluated by FE-SEM and confocal images aer b ft eing cultured for 3 days (Fig. 6A–D). Compared to the randomly oriented nanob fi rous sample, the FE-SEM image of the radially aligned nanofibrous sample shows a better cell proliferation rate. The confocal image also shows a higher cell proliferation rate in radially aligned nanob fi rous scao ff lds, further confirming the orientation of the rCCs. The proliferation of corneal cells on the matrixes for transplantation is important for the recovery of 36,39 vision . The proliferation of corneal cells in a different topography of the nanob fi rous scao ff lds was evaluated using CCK-8 assay aer 1, 3, a ft nd 5 days of cell seeding (Fig. 6E). No remarkable difference is observed between the rates of cell proliferation among the experimental groups on day 1. However, the radially aligned nanob fi rous sam- ple shows a significant increase compared to the randomly oriented nanob fi rous sample aer 3 d ft ays of cell culture. Proliferation results of the FE-SEM, confocal images, and CCK-8 assay suggest that the orientation of nanob fi ers in the fabricated scao ff ld could play an important role in the behavior of corneal cells or anisotropic tissue. e Th 3D radially oriented nanob fi rous scao ff lds showed excellent ability to induce cell morphogenesis and cell migration as well as mechanical properties. The degree of alignment of the nanob fi ers also ae ff cted the size of the rCCs. On the randomly oriented nanob fi rous mat, rCCs with the size of 10–20 um were the most common, and on the aligned nanob fi rous mat, the spherically shaped rCCs smaller than 10 um were the most common (Fig. 6F). On the aligned nanob fi rous mat, however, elongated cells larger than 50 um and smaller than 100 um were observed. es Th e results appear to be due to the difference in pore size and alignment among the nanob fi rous mats. e a Th mount of cell DNA on the radially aligned nanob fi rous 3D mats was higher than that on the randomly oriented nanob fi rous mats (Fig. 6G). The amount of cell DNA on the control polystyrene dishes was significantly lower than that on the proposed hemispherical 3D nanob fi rous scao ff lds with radially aligned patterns. rCCs migration. The cells are surrounded by the ECM, the complex network consisting of molecules, pro- teins, and polysaccharides. The cells in the native tissues migrate in response to various gradients of stimula- 13,40 41 tion . The regulation of the cell migration is of paramount importance in biomedical application field . One effective way is a creation of a microenvironment which mimics the target tissue complexity by incorporating bio- logical and physical gradients into a scao ff ld. Topographical cues like porosity, pore size, alignment, and stiffness of the scao ff ld can be crucial for cell migration and behavior, inducing cell polarity and controlling the cellular activities and proliferation rate . The proposed hemispherical 3D nanofibrous scaffolds with radially aligned patterns in the manuscript not only have aligned topographic features like aligned nanob fi rous mat but also have gradients of stimuli such as porosity and pore size that can ae ff ct the polarity and migration rate of the cells. The SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 8 www.nature.com/scientificreports/ Figure 6. (A) SEM image of corneal cells of a rabbit attached aer 3 d ft ays of culture on a randomly oriented nanob fi rous mat. ( B) Confocal microscopy images of corneal cells of a rabbit attached aer 3 d ft ays of culture on a randomly oriented and aligned nanob fi rous mat. (C) SEM image of corneal cells of a rabbit attached aer ft 3 days of culture on an aligned nanob fi rous mat. (D) Confocal microscopy images of corneal cells of a rabbit attached aer 3 d ft ays of culture on an aligned nanob fi rous mat. Actin Green 488 (green) was applied for actin filament and DAPI (blue) for staining nuclei. (E) CCK-8 assay result of corneal cells of rabbit on random and radially aligned nanob fi rous mats aer 1, 3, a ft nd 5 days of cell culture. (F) Distribution of cell size for corneal cells of a rabbit attached aer 3 d ft ays of culture on randomly oriented and aligned nanob fi rous mats. (G) Pico Green dsDNA quantification assay. SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 9 www.nature.com/scientificreports/ Figure 7. (A,B) Fluorescence images comparing the cell migration when rCCs were cultured on the membranes of randomly oriented and radially aligned fibers, respectively, for 8 days. The scratch test adapted to the mats with a 5 × 3 × 3mm stainless steel strip for cell migration studies along their surface topography. constructs developed in this study have the advantages (including a transparency) of both ordered nanofibers and gradient materials. This is also shown in other papers about the fabrication of the radially oriented fibrous constructs. Xie et al. have demonstrated the method of manufacturing an electrospun fibrous structure consist - ing of radially aligned PCL fibers to mimic the dura mater and to enhance the movement of the cells from the surrounding tissue to the center of dural defects . Their scao ff lds based on radially aligned fibers showed great potential as dural scao ff lds to induce wound healing and regeneration. To evaluate cell migration and motility on this membrane, rCCs were stained with actin green and fluores- cence images were taken at different times. The scratch test was adapted to the membranes with a stainless steel strip along their surface on day 3 aer ft seeding. Figure 7 showed rCCs distribution ae ft r seeding on the scao ff lds of random and radially aligned fibers on day 3, 4, 5, 6, and 8 day. The ability for rCCs to repopulate the simu- lated defect was measured for cell migration assay. The void area decreased with increasing culture time for the membranes because of the inward migration of rCCs. The hemispherical 3D nanob fi rous scao ff lds with radially aligned patterns can significantly promote migration of cells when compared to randomly oriented 2D fibrous mats. Even aer 6 ft day of scratching on the random fibrous membrane, the rCCs showed a residual bare surface of about 4mm as shown in Fig. 7A. However, the cells migrated and recovered the entire portion of the defect aer ft 5 day of culture time for the radially aligned fibrous membrane (Fig. 7B). Immunohistochemical analysis. Immunohistochemical analysis results with ZO-1 staining of the scaf- folds with rCCs demonstrate that the rCCs and the matrixes were attached with their retained function as shown in Fig. 8. The relative quantity of zona ocludin-1 (ZO-1) in the corneal cells is important to the properties of the mats for cornea regeneration because ZO-1 determines the distribution of F-action and functions as a major 44,45 cytoskeletal organizer in endothelial cells . The expression of ZO-1 of the rCCs on the radially aligned nanofi- brous scao ff lds shows a much higher intensity of staining and denser stains than on the randomly oriented nano- b fi rous scao ff lds. Moreover, the cultured rCCs on the ordered nanob fi rous matrixes show the better organization of ZO-1. mRNA Expression. To confirm the expression of m RNA, gene markers for rCCs such as ZO-1, Na+ / K+-ATPase (NaK), and CLCN3 were used as shown in Fig. 8I. All gene markers were normalized by Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). CLCN3 that plays the role of regulator of pH, prolifera- tion, and immigration of cell-to-cell is important in keeping the proper size and morphology of rCCs. NaK that is an enzyme found in cell membrane facilitates maintenance of the transparency of cornea through control of the edema of pump function. Compared with that of the randomly oriented fibrous membrane, most of the genes showed a higher expression rate in the radially oriented fibrous membrane. The rCCs on the hemispherical 3D nanofibrous scao ff lds with radially aligned patterns showed highly enhanced expression of ZO-1 and CLCN3. Compared to the control group (TCP), electrospun fibrous membranes have favorable environments for typi- cal gene expression of rCCs, especially 3D nanofibrous scao ff lds with radially aligned patterns, and provide for prominent roles in cell proliferation and adhesion. As a result, the 3D nanob fi rous scao ff lds with radially aligned patterns show great potential as artificial corneal substrates and may open novel avenues for clinical carriers with maintaining the phenotype of rCCs. Conclusions We designed a new electrospinning setup to enable the production of a hemispherical 3D nanofibrous scao ff ld consisting of radially aligned nanofibers that mimic the isotropic tissues that grow in a directional orienta- tion. Unlike current electrospinning methods, the developed electrospinning method is capable of producing SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 10 www.nature.com/scientificreports/ Figure 8. (A,B) Immunouo fl rescent staining of ZO-1 in the corneal cells aer 3 a ft nd 7 days culture on the PCL random nanob fi rous scao ff lds. (C,D) Immunouo fl rescent staining of ZO-1 in the corneal cells aer t ft he 3 and 7 days culture on the PCL aligned nanob fi rous scao ff lds. (E,F) Immunouo fl rescent staining of ZO-1 in the corneal cells aer t ft he 3 and 7 days culture on the random PCL/collagen nanob fi rous scao ff lds. (G,H) Immunouo fl rescent staining of ZO-1 in the corneal cells aer t ft he 3 and 7 days culture on the aligned PCL/ collagen nanob fi rous scao ff lds (DAPI stained nuclei of the corneal cells were used for normalization). (I) Specific gene expression of rCCs by RT-PCR (normalized by GAPDH). hemispherical scao ff lds such as contact lenses, and is designed for application to curved tissues such as those of the eye, elbow, or 3D wound area. 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Understanding the process of corneal endothelial morphological change in vitro. Invest Ophthalmol Vis Sci 56, 1228–1237, https://doi.org/10.1167/iovs.14-16166 (2015). 45. Benezra, M., Greenberg, R. S. & Masur, S. K. Localization of ZO-1 in the nucleolus of corneal fibroblasts. Invest Ophthalmol Vis Sci 48, 2043–2049, https://doi.org/10.1167/iovs.06-0754 (2007). SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 12 www.nature.com/scientificreports/ Acknowledgements This research was supported by grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) by Ministry of Education, Science and Technology (Project no. 2016R1A2A2A07005160), and the program for fostering next-generation researchers in engineering of National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT (2017H1D8A2030449). Author Contributions All experiments and manuscript preparation are designed by first author (J.I. Kim). J.I. Kim, performed the experiment and wrote the main manuscript text. J.Y. Kim, analyzed electricfield using COMSOL software. C.H. Park, supervised all of manuscript. Additional Information Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCiENtifiC REPO R ts | (2018) 8:3424 | DOI:10.1038/s41598-018-21618-0 13
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